The aims of this R03 proposal are to perform the initial experiments: 1. to develop a system of gene transfer from Salmonella to eukaryotic host cells; 2. to examine the Salmonella properties and host cell factors that affect gene transfer processes; and 3. compare conjugation with other Salmonella-based DNA transfer systems. The first aim emerges from our recent discovery of E. coli-to-mammalian cell conjugative DNA transfer. The methodology developed for the E. coli system will be adapted to donor strains of Salmonella, in order to ultimately capitalize on bacterial-host cell interactions that are unique to the Salmonella organism. With the noninvasive E. coli K12 donors, non-conjugative mechanisms of DNA transfer such as transformation and bacterial engulfment were conclusively ruled out by rigorous controls, including the use of Dnasel in the milieu and inhibitors of endocytosis. With the Salmonella system, the hypothesis is that more than one DNA delivery mechanism will be operative, since Salmonella has multiple ways of interacting with host cells. Such delivery mechanisms could involve intracellular ("intracytoplasmic") bacterial delivery, extracellular bacterial delivery, and intravacuole bacterial delivery. In each cellular locale, distinct processes can be at work. The second aim will help define the bacterial and host cell properties that impact these events. This approach will characterize the transfer mechanisms while providing ways to improve DNA delivery as a therapeutic tool. The third aim will use those defining factors to distinguish bacterial conjugative transfer by Salmonella from that of E. coli and to compare conjugative transfer with the other Salmonella-based DNA transfer systems. These studies are intended to develop attenuated Salmonella strains for DNA delivery for DNA vaccine approaches, corrective gene therapy, immunotherapy, and the prevention and treatment of certain kinds of cancer.